| AIM: To develop and validate a rapid and sensitive liquid chromatography- tandem mass spectrometry (LC-MS/MS) assay for determination of cisapride in human plasma. Then apply this assay to study the bioequivalence of cisapride in table formulation in healthy volunteers given an oral dose (10 mg)STUDY METHOD: Cisapride and the internal standard (I.S.) diphenhydramine were extracted from human plasma using diethyl ether-dichloromethane (60:40, v/v), and then separated on a Venusil Mp C18 (4.6×150 mm I.D., 5μm ) column using methanol-10 mM ammonium acetate-formic acid (65:35:0.035 v/v/v). The mass spectrometric detection took place in the MS-MS mode with ionization of the analyte in an ESI ion source working in the positive mode. Using multiple reaction monitoring (MRM), the transitions m/z 467.1→184.1 and m/z 256.1→167.1 were used for quantitation of cisapride and diphenhydramine (I.S.), respectively. The linearity, specificity, LLOQ, precision, accuracy, recovery and stability were estimated in the method of validation for human plasma.The method was applied to evaluate the bioequivalence of two tablets of cisapride in 22 healthy male healthy volunteers (10 mg). The blood samples (about 4 mL) were collected in heparinized tubes at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 8.0, 10, 12, 24 and 36 h after oral administration. All blood samples were separated following centrifugation at 3000×g for 10 min and the plasma samples were stored at ?20°C until analysis.The concentrations of cisapride in health volunteers'plasma samples were measured. The pharmacokinetic parameters were derived from the plasma concentration–time profile. Based on these, the bioavailability of two tablet formulations was estimated by a 90% confidence intervals and a two one-sided t-test of test to reference ratio (after log-transformed) of the AUC0-t, AUC0-∞and that of Cmax (after log-transformed).RESULTS: A rapid and sensitive LC-MS/MS method has been developed and validated for the determination of cisapride in human plasma. Linearity was established for the range of concentrations 0.3?100 ng/mL with a coefficient of determination (r) of 0.9989. The limit of quantitation (LLOQ) was 0.3 ng/mL. In the range of the standard curve, there is no co-eluting endogenous substances significantly influenced the determination of flunarizine or I.S. at the retention times in human plasma.The precisions (R.S.D., %) were all less than 4.52% and the accuracy (R.E., %) was within±3.43%. Long-term stability was studied by assaying samples after storage for one month at ?20°C. The freeze-thaw stability was evaluated by analyzing samples after undergoing three-thaw cycles. To test the post-preparative stability, the prepared samples were placed into the autosampler at room temperature for 6 h. Cisapride had an acceptable stability at ?20°C for 1 month, in the autosampler at room temperature for 6 h after protein precipitation and after three freeze-thaw cycles. Thus, no stability-related problems are expected during the routine analyses for our study. In summary, this assay fully meets the conformance to relevant standards of Pharmacopoeia of People's Republic of China, and applied to bioequivalence studies of cisapride tablets in clinical trials.The assay was applied to evaluate the bioequivalence of two tablet formulations of cisapride in 22 healthy adult male volunteers who received a single dose (containing 10 mg cisapride) in a two-period randomized crossover design with a one-week washout period between doses. After the concentrations of cisapride in health volunteers'plasma samples were measured, the mean plasma concentration–time curves for two tablets were obtained, and the pharmacokinetic parameters were derived from these curves. Calculated from mean AUC0-t, the relative bioavailability of the test formulation was 103.4±37.9%.There were no significant differences between the two formulations by a two one-sided t-test (P>0.05). The 90% confidence intervals of test to reference ratio (after log-transformed) of Cmax (after log-transformed) (88.78– 114.08%) for cisapride, were within 70– 143%, and that of. the AUC0-t (86.59– 110.78%) and AUC0-∞(85.98– 109.38%) for cisapride, were within the bioequivalence criteria range of 80– 125%.The test formulation and reference formulations were therefore considered to be bioequivalent.
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